Method for producing dihydroxyacetone-3-phosphate

ABSTRACT

Methods are provided to produce highly concentrated dihydroxyacetone-3-phosphate efficiently by a simple catalytic reaction of dihydroxyacetone with the bacterial cells transformed with the gene encoding a dihydroxyacetone kinase or the dihydroxyacetone kinase produced by said bacterium.

TECHNICAL FIELD

The present invention relates to a method for producingdihydroxyacetone-3-phosphate using enzyme.

BACKGROUND ART

Dihydroxyacetone-3-phosphate is a substrate for fructose1,6-bisphosphate aldolase and is used for stereoselectively synthesizingmany kinds of carbohydrates by aldol condensation with variousaldehydes. The carbohydrates thus synthesized can be widely utilized asmedicines and their synthetic intermediates. The above reaction,however, requires large excesses of expensivedihydroxyacetone-3-phosphate, making the products expensive.

For example, dihydroxyacetone-3-phosphate can be chemically produced bydirectly phosphorylating dihydroxyacetone dimer with phosphorusoxychloride (POCl₃) in pyridine (Tetrahedron Lett., 28, 1641 (1987)).This production method is complicated and thus cannot producedihydroxyacetone-3-phosphate in a high yield at low cost.

Methods of producing dihydroxyacetone-3-phosphate using enzymes havealso been reported. An example thereof is a method of synthesizingdihydroxyacetone-3-phosphate from dihydroxyacetone with immobilizedglycerol kinase (GK) under conditions that ATP is regenerated by usingphosphoenolpyruvic acid and pyruvate kinase (PK) (J. Org. Chem., 48,3199(1983), and J. Am. Chem. Soc., 107, 7019(1985)). In this method,acetate kinase and acetylphosphate may be used instead ofphosphoenolpyruvic acid and pyruvate kinase (J. Am. Chem. Soc., 107,7019(1985)). These methods using glycerol kinase (GK) require highlypurified enzymes and therefore cannot producedihydroxyacetone-3-phosphate in a high yield at low cost.

For another example, it is theoretically possible to producedihydroxyacetone-3-phosphate from dihydroxyacetone usingdihydroxyacetone kinase (DHAK) (examined published Japanese patentapplications (JP-B) No. Hei 4-29349 and Hei 4-22560). In particular,dihydroxyacetone kinase derived from yeast Schizosaccharomyces pombe issuitable for producing dihydroxyacetone-3-phosphate (JP-B Hei 4-29349).However, the cell extract of Schizosaccharomyces pombe containscoexisting enzymes such as phosphatases and triose-3-phosphateisomerase, which degrade the formed dihydroxyacetone-3-phosphate. Theenzyme thus needs to be highly purified and has not yet been appliedpractically.

For the practical production of dihydroxyacetone-3-phosphate usingenzymes, the enzyme or the biocatalyst containing the enzyme should havehigh reactivity, be prepared easily at low cost, and be highly purifiedso as not to degrade the product, dihydroxyacetone-3-phosphate. Noefficient method for producing dihydroxyacetone-3-phosphate by usingenzymes or biocatalysts containing the enzymes, which meets the aboverequirements, has been developed.

DISCLOSURE OF THE INVENTION

An objective of this invention is to provide a method, for efficientlyproducing dihydroxyacetone-3-phosphate.

Under the above circumstances, the present inventors have attempted toclone a gene encoding dihydroxyacetone kinase and to express itefficiently to obtain the practical enzyme. The present inventors havesucceeded in overexpressing the enzyme in a microbial host. Furthermore,the present inventors have found that dihydroxyacetone-3-phosphate canbe produced in a high concentration by contacting the host cells as theyare or the enzyme produced by them with dihydroxyacetone, the substrateof the enzyme, thereby accomplishing the present invention.

More specifically, this present invention relates to:

(1) a method for producing dihydroxyacetone-3-phosphate, which comprisescontacting dihydroxyacetone with bacterial cells transformed with thegene encoding dihydroxyacetone kinase or the enzyme produced by thebacterial cells;

(2) a method described in (1), wherein said dihydroxyacetone kinase isderived from yeast belonging to the genus Schizosaccharomyces;

(3) a method described in (1), wherein said dihydroxyacetone kinase isderived from Schizosaccharomyces pombe;

(4) a method described in (1), wherein said bacterial cell isEscherichia coli; and

(5) a method described in any one of (1) to (4), wherein said contactingis performed under the conditions that coenzyme, ATP, is regenerated.

The source of the gene encoding dihydroxyacetone kinase used in thepresent invention is not limited. It is preferably derived from yeastbelonging to the genus Schizosaccharomyces; more preferably,Schizosaccharomyces pombe; and still more preferably,Schizosaccharomyces pombe IFO 0354.The nucleotide sequence of thedihydroxyacetone kinase gene from Schizosaccharomyces pombe IFO 0354 isshown in SEQ ID NO: 1, and the deduced amino acid sequence is shown inSEQ ID NO: 2.

In the method of this invention, microorganisms transformed withdihydroxyacetone kinase capable of overexpressing the enzyme can be usedas they are as an enzyme catalyst. The term “overexpression” as usedherein means a higher level expression than spontaneous |expression. Ingeneral, transformants capable of overexpresing the expression productcan be obtained by introducing multiple copies of the expression unitwith the gene encoding the expression product into host cells. The hostbacterial cells used in the present invention are not particularlylimited. It is preferable to use bacteria belonging to the genusEscherichia, and more preferable to use E. coli K-12 strain becausevarious host-vector systems can be used. For example, pUC118 or pUC119is the preferable expression vector into which the dihydroxyacetonekinase gene is to be inserted (Methods in Enzymology, 153, 3 (1987)).The gene can be introduced into the host cell by methods well known inthe art such as a method described in “Methods in Enzymology, 68, 299(1979).” For example, E. coli capable of overexpressing dihydroxyacetonekinase can be prepared as described below. First, the primers aredesigned for polymerase chain reaction (PCR) based on the databaseresulting from genome analysis of Schizosaccharomyces pombe. The targetDNA is amplified by PCR using the primers and genomic DNA isolated fromSchizosaccharomyces pombe IFO 0354 as a template DNA by conventionalprocedures. The PCR products are ligated into appropriate vectors andare cloned. The gene-inserted vectors are transformed into anappropriate strain of E. coli, and the transformants are then selectedby detecting the existence of drug resistant gene(s) and the DNAinsert(s). The DHAK productivity of the clones is measured by assayingthe activity of the enzyme.

The enzyme produced by the bacterial cells can be isolated from thecells for use in the method of the present invention. The enzyme is notnecessarily purified. A crude enzyme is used as well as the purifiedenzyme. The dihydroxyacetone kinase can be recovered from the growncells by extracting the enzyme from the cells first because the enzymeis produced intracellularly. Namely, the cells are collected from theculture medium by filtration or centrifugation and disrupted bymechanical methods such as treatment with alumina, dynomill, orultrasonication to extract the enzyme in a soluble form. Alternatively,the cell membranes are destroyed by treatment with organic solvents suchas acetone, and the resulting cells are dried under reduced pressure.The powder thus prepared is used as a catalyst containing the enzyme.Insoluble materials are removed from the culture obtained in the methodsdescribed above by filtration or centrifugation to obtain the crudeenzyme. Furthermore, the crude enzyme is concentrated and purified bymethods known in the art such as adsorption chromatography, ion exchangechromatography, and gel filtration chromatography. The thus-obtained,partially purified preparation of dihydroxyacetone kinase is also usedin the method of this invention.

The dihydroxyacetone kinase activity of the E. coli strain thatoverexpresses the enzyme or the enzyme isolated from said cells ismeasured by known methods (JP-B Hei 04-29349) as follows. The decreaseof absorbance at 340 nm is spectrophotometrically measured during thereaction at 25° C. in 1 ml of the reaction mixture containing 0.1Mtriethanolamine buffer (pH 7.5), 2.5 mM ATP, 4 mM MgSO₄, 0.2 mM NADH,2.5 units of glycerol-3-phosphate dehydrogenase (G3PDH), and 0.01 ml ofa test enzyme solution (formula below).

DHAK DHA + ATP → DHAP + ADP G3PDH DHAP + NADH → Glycerol-3-phosphate +NAD⁺

One unit of the enzyme of the present invention is defined as the amountrequired to decrease 1 μmole of NADH in 1 min under the conditionsdescribed above.

Dihydroxyacetone-3-phosphate is usually produced by the method of thisinvention under the following conditions. First, the reaction is carriedout in a buffer with pH ranging from 7 to 8. Any buffer solution can beused as long as it can keep its pH within the above reaction pH range.Tris-hydrochloride buffer is preferable. The reaction temperature may beany range as long as dihydroxyacetone kinase used is active. Preferably,the temperature ranges from about 20° C. to about 35° C. It is necessaryto add ATP to the reaction mixture since dihydroxyacetone kinaserequires ATP as a coenzyme. However, the enzyme is inhibited by theformed ADP. Therefore, the reaction is preferably performed withregenerating ATP. Any known systems for regenerating ATP (J. Org. Chem.48, 3199 (1983) and J. Am. Chem. Soc. 111, 627 (1989)) can be used. Inview of the production cost, a system using acetyl phosphate and acetatekinase is preferred.

Dihydroxyacetone-3-phosphate produced by the above reaction can beidentified by enzymatic and chemical techniques. Namely, the decrease ofabsorbance at 340 nm is spectrophotometrically measured during thereaction (formula below) at 30° C. in 3 ml of a reaction mixturecontaining 0.1 M sodium acetate buffer (pH 6.0), 0.25 mM NADH, 0.6 unitsof glycerol-3-phosphate dehydrogenase (G3PDH), and 0.01 ml of a testsolution. The concentration of dihydroxyacetone-3-phosphate (DHAphosphate) in the test solution is then determined based on acalibration curve which is made in advance using the standard compound.

G3PDH DHAP + NADH → Glycerol-3-phosphate + NAD⁺

After the above reaction, the reaction product can be precipitated byadding organic solvents such as ethanol to the reaction mixture, or bysalting out, to recover it. Furthermore, the reaction product ispurified by usual purification procedures such as column chromatographyand recrystallization. Alternatively, it can be subjected to the nextprocedure as it is.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of the pUDK vector.

BEST MODE FOR IMPLEMENTING THE INVENTION

The following examples illustrate the present invention in more detail,but are not to be construed to limit the scope of the present invention.

EXAMPLE 1 Preparing Escherichia coli Strain OverexpressingDihydroxyacetone Kinase

The DHAK gene derived from Schizosaccharomyces pombe IFO 0354 wasobtained and analyzed in the following manner. Two primers, primer A(5′-GCCGGTACCAGGAGCTAAAATATGGATAAGCAC-3′, SEQ ID NO: 3) and primer B(5′-GCCAAGCTTAAAATTTCGTATCCAATTATTCG-3′, SEQ ID NO: 4) were designedbased on the database resulting from the genome analysis ofSchizosaccharomyces pombe. Primer A contains the restrictionendonuclease KpnI recognition site and E. coli SD sequence, while primerB contains the restriction endonuclease HindIII recognition site. Thetarget gene (dhakI) was amplified by PCR using genomic DNA isolated fromSchizosaccharomyces pombe IFO 0354 by a conventional method as atemplate DNA and the above primers. The PCR products were ligated withpT7BlueT vectors and cloned. The coding region of the desired gene wasthen isolated from the vector, and ligated with pUC118 vector. Thevector thus obtained was named pUDK.

E. coli strain JM109 was transformed with pUDK. The transformant thusobtained was named “E. coli JM109 (pUDK).” Dihydroxyacetone kinase wasproduced by this transformant as follows. E. coli JM109 (pUDK) wasinoculated into LB broth (1% tryptone, 0.5% yeast extract, 1% NaCl, pH7.0) containing 100 mg/ml of ampicillin, 0.4 mm IPTG(isopropylthiogalactopyranoside) as an inducer and cultured at 37° C.for 16 hours with shaking. FIG. 1 shows the structure of the pUDKvector. SEQ ID NO: 1 shows the nucleotide sequence of dhakI, and SEQ IDNO: 2 shows the deduced amino acid sequence. The molecular weight ofthis enzyme was estimated to be 145,000 by an HPLC analysis, and themolecular weight of the subunit was estimated to be 63,000 bySDS-polyacrylamide gel electrophoresis. The molecular weight of thesubunit agreed well with the molecular weight (62,245) of the deducedamino acid sequence. The requirement of divalent metal cations on theenzyme activity was investigated. The relative activity of the enzyme inthe presence of Ca²⁺, Co²⁺, and Mn²⁺ is 116%, 20%, and 12%,respectively, taking the relative activity of the enzyme in the presenceof Mg²⁺ as 100%. These results indicate that this enzyme is dimeric andthat the cloned dhakI gene encodes the isozyme of dihydroxyacetonekinase I derived from Schizosaccharomyces pombe (JP-B Hei 04-29349).

EXAMPLE 2 Comparison of the Activities of Crude Enzyme Solutions Derivedfrom E. coli JM109 (pUDK) and S. Pombe

E. coli JM109 (pUDK) was inoculated into LB broth (1% trypton, 0.5%yeast extract, 1% NaCl, pH7.0) containing 0.4 mM IPTG(isopropylthiogalactoside) and cultured at 37° C. for 16 hours withshaking. The cells were collected from the culture medium bycentrifugation and resuspended in a buffer solution. The suspended cellswere disrupted by sonication, and the lysate was then centrifuged toobtain the supernatant as a crude enzyme solution. Schizosaccharomycespombe IFO 0354 was cultured according to the method described in JP-BHei 04-29349, and the collected cells were disrupted by aluminum oxideto obtain the crude enzyme solution in the same manner as above. Theactivities (unit/dl of broth) of dihydroxyacetone kinases andcontaminating enzymes from both strains are shown in Table 1.

TABLE 1 Schizosaccharomyces E. coli JM109 (pUDK) pombe IFO 0354 DHAK 8014 alkaline phosphatase 0 2.2 triose-3-phoshate 58 485 isomerase

As clearly shown in Table 1,the crude enzyme solution from E. coli JM109(pUDK) has high dihydroxyacetone kinase activity and low activities ofcontaminating enzymes, while the crude enzyme solution from S. pombe IFO0354 has low dihydroxy kinase activity and high activities ofcontaminating enzymes. The productivity of dihydroxyactone-3-phosphateusing the crude enzyme solution from both strains was measured byallowing 10 ml of a reaction mixture containing 100 mM DHA, 10 mM MgSO₄,4 mM ATP, 100 mM acetyl phosphate (AcOP), 6 units of AK (Unitika Co.,Ltd), 6 units of DHAK, and 0.1M Tris-HCl buffer (pH 7.3) to react at 25°C. for 10 to 15 hours. As a result, a significant difference wasobserved in the two. The results are shown in Table 2.

TABLE 2 DHAK concentration (mM) Time (hours) E.coli JM109 (pUDK) S.pombe IFO 0354 0 0 0 1 32 7 2 41 4 4 63 4 6 70 4 8 63 3 24 55 4

EXAMPLE 3 Producing Dihydroxyacetone-3-phosphate Using a Crude EnzymeSolution from E. coli JM109 (pUDK)

Ten milliliters of a reaction mixture containing 50 mM DHA, 10 mM MgSO₄,60 mM ATP, 6 units of dihydroxyacetone kinase, and 0.1 M Tris-HCl buffer(pH 7.3) was allowed to react at 25° C. for 10 to 20 hours to produce 20mM DHA phosphate (3.8 mg/ml of monosodium dihydroxyacetone-3-phosphate).

EXAMPLE 4 Producing Dihydroxyacetone-3-phosphate Using a Crude EnzymeSolution from E. coli JM109 (pUDK) with an ATP Regeneration System

Ten milliters of a reaction mixture containing 100 mM DHA, 10 mM MgSO₄,4 mM ATP, 100 mM acetyl phosphate (AcOP), 6 units of AK (Unitika Co.,Ltd), 6 units of a crude DHAK solution, and 0.1M Tris-HCl buffer (pH7.3) was allowed to react at 25° C. for 10 to 15 hours, producing 70 mMDHA phosphate (13.3 mg/ml of monosodium dihydroxyacetone-3-phosphate).

EXAMPLE 5 Producing Dihydroxyacetone-3-phosphate Using E. coli JM109(pUDK) with an ATP Regeneration System

The reaction was performed in the same manner as in Example 2 except forusing E. coli JM109 (PUDK) cells containing 6 units of dihydroxyacetonekinase and produced 73 mM DHA phosphate (13.9 mg/ml of monosodiumdihydroxyacetone-3-phosphate).

EXAMPLE 6

Ten milliliters of a reaction mixture containing 200 mM DHA, 10 mMMgSO₄, 4 mM ATP, 250 mM acetyl phosphate (AcOP), 24 units of AK (UnitikaCo., Ltd), 24 units of a crude DHAK solution, and 0.1 M Tris-HCl buffer(pH 7.3) was allowed to react at 25° C. for 15 to 20 hours whilecontrolling the pH; 140 mM DHA phosphate (26.6 mg/ml as monosodiumdihydroxyacetone-3-phosphate) was then produced. The time course of thereaction is shown in Table 3.

TABLE 3 Time (hours) DHAP concentration (mM) 0 0 1 35 2 57 4 81 6 102 8116 10 133 12 140 24 116

The reaction mixture was treated with active carbon and filtrated.Barium acetate (0.08 g) was then added to the filtrate, and theresulting precipitate was removed by filtration. The filtrate wasconcentrated to 3 ml under reduced pressure, then a five-fold volume ofcold ethanol was added to the filtrate. The resulting precipitate wasfiltered and dried under reduced pressure to obtain 180 mg of monosodiumdihydroxyacetone-3-phosphate.

INDUSTRIAL APPLICABILITY

The present invention provides a method for efficiently producingdihydroxyacetone-3-phosphate in a high concentration by using theenzymatic reaction of dihydroxyacetone kinase.

4 1 1743 DNA Schizosaccharomyces pombe CDS (1)..(1743) IFO 0354 1 atggat aag cac ttt atc aac gat cct gaa gtc ctc gtc ctt gat ggc 48 Met AspLys His Phe Ile Asn Asp Pro Glu Val Leu Val Leu Asp Gly 1 5 10 15 cttaaa tcc ttg gcc gac atg aac aaa act tta act gtt cat gaa gag 96 Leu LysSer Leu Ala Asp Met Asn Lys Thr Leu Thr Val His Glu Glu 20 25 30 gga aaattc atc tat ttc cat gac tac aac aaa aag aat gtc agt gtc 144 Gly Lys PheIle Tyr Phe His Asp Tyr Asn Lys Lys Asn Val Ser Val 35 40 45 att tcc ggcggt ggt gct ggt cat gaa ccc act cat tct tcg ttc gtg 192 Ile Ser Gly GlyGly Ala Gly His Glu Pro Thr His Ser Ser Phe Val 50 55 60 ggc aag ggt atgctt act gcc gcc gtc tca ggc tcc att ttt gct tct 240 Gly Lys Gly Met LeuThr Ala Ala Val Ser Gly Ser Ile Phe Ala Ser 65 70 75 80 ccg tcg tca aagcaa att tat acc ggt att aag caa gtc gaa tct gag 288 Pro Ser Ser Lys GlnIle Tyr Thr Gly Ile Lys Gln Val Glu Ser Glu 85 90 95 gct ggc acc ttg gtaatt tgc aaa aac tac acc ggt gac atc ctt cac 336 Ala Gly Thr Leu Val IleCys Lys Asn Tyr Thr Gly Asp Ile Leu His 100 105 110 ttt ggt atg gcc ttggag aag caa aga acg gct ggt aag aag gct gaa 384 Phe Gly Met Ala Leu GluLys Gln Arg Thr Ala Gly Lys Lys Ala Glu 115 120 125 ctt att gcc gtt gcagat gac gta tca gta ggt cgt aag aag agc ggt 432 Leu Ile Ala Val Ala AspAsp Val Ser Val Gly Arg Lys Lys Ser Gly 130 135 140 aag gtc gga cgt cgtggt ttg tct ggt act gtt ctt gtt cac aaa atc 480 Lys Val Gly Arg Arg GlyLeu Ser Gly Thr Val Leu Val His Lys Ile 145 150 155 160 gct ggt gca gctgcc gcc aga ggg tta cct ttg gaa gcc gtt acg acc 528 Ala Gly Ala Ala AlaAla Arg Gly Leu Pro Leu Glu Ala Val Thr Thr 165 170 175 att gct aag gctgct att gac aat ttg gtt agt atc ggt gct tca ctc 576 Ile Ala Lys Ala AlaIle Asp Asn Leu Val Ser Ile Gly Ala Ser Leu 180 185 190 gct cac gtt cacgtc cct ggt cat gag cca att gca aaa gaa gat gaa 624 Ala His Val His ValPro Gly His Glu Pro Ile Ala Lys Glu Asp Glu 195 200 205 atg aaa cat gatgaa atg gaa ctt gga atg ggt att cac aat gaa cct 672 Met Lys His Asp GluMet Glu Leu Gly Met Gly Ile His Asn Glu Pro 210 215 220 gga tgc aag cgtatt tcc cct att ccc tct att gat gac cta att gct 720 Gly Cys Lys Arg IleSer Pro Ile Pro Ser Ile Asp Asp Leu Ile Ala 225 230 235 240 cag atg cttaag caa atg ttg gat caa tcc gac aag gac cgt gcc tat 768 Gln Met Leu LysGln Met Leu Asp Gln Ser Asp Lys Asp Arg Ala Tyr 245 250 255 gta aag attgag ggt gac gat gaa gta gtc tta ctt atg aat aac ctt 816 Val Lys Ile GluGly Asp Asp Glu Val Val Leu Leu Met Asn Asn Leu 260 265 270 ggt ggt ctttcc atg ctt gaa ttc agt gcc att agc cac aag gtg aag 864 Gly Gly Leu SerMet Leu Glu Phe Ser Ala Ile Ser His Lys Val Lys 275 280 285 gaa gca ttggct aaa gaa tac aaa atc aac ccc gtt cgc atc ttt gcc 912 Glu Ala Leu AlaLys Glu Tyr Lys Ile Asn Pro Val Arg Ile Phe Ala 290 295 300 ggt cca tttacc acc agt ttg aat ggc ttg ggt ttc ggt atc act ttg 960 Gly Pro Phe ThrThr Ser Leu Asn Gly Leu Gly Phe Gly Ile Thr Leu 305 310 315 320 ctc cgtacc act gac cgc gtc aaa gtc gag ggc gaa gaa tac tct ttg 1008 Leu Arg ThrThr Asp Arg Val Lys Val Glu Gly Glu Glu Tyr Ser Leu 325 330 335 gtt gatttg att gac caa cct gtt gaa gct atc gga tgg cct ttg tgt 1056 Val Asp LeuIle Asp Gln Pro Val Glu Ala Ile Gly Trp Pro Leu Cys 340 345 350 caa ccctct gac ttg aag tcc aaa aac aag att ggc aat gtc agc atc 1104 Gln Pro SerAsp Leu Lys Ser Lys Asn Lys Ile Gly Asn Val Ser Ile 355 360 365 gag gagggt cag aag gat gtc aag tct ccc gtt act gtc gat aag gag 1152 Glu Glu GlyGln Lys Asp Val Lys Ser Pro Val Thr Val Asp Lys Glu 370 375 380 aag gttcgt cag gcg att gtc aat tcg atg gag aat ctc atc aaa gca 1200 Lys Val ArgGln Ala Ile Val Asn Ser Met Glu Asn Leu Ile Lys Ala 385 390 395 400 gagcct aaa att aca aag ttc gat acg atg gct ggt gat ggt gac tgt 1248 Glu ProLys Ile Thr Lys Phe Asp Thr Met Ala Gly Asp Gly Asp Cys 405 410 415 ggtact act ttg aag cgt ggt gct gaa ggt gtt ttg aag ttt gtt aaa 1296 Gly ThrThr Leu Lys Arg Gly Ala Glu Gly Val Leu Lys Phe Val Lys 420 425 430 tccgac aaa ttc tct gac gat cct att cgt att gtt cgt gat atc gca 1344 Ser AspLys Phe Ser Asp Asp Pro Ile Arg Ile Val Arg Asp Ile Ala 435 440 445 gatgtt att gaa gac aat atg gat ggt aca tct ggt gct ttg tac gcc 1392 Asp ValIle Glu Asp Asn Met Asp Gly Thr Ser Gly Ala Leu Tyr Ala 450 455 460 attttc ttc cat ggc ttt gcg aag ggc atg aaa gac acc ttg gag aag 1440 Ile PhePhe His Gly Phe Ala Lys Gly Met Lys Asp Thr Leu Glu Lys 465 470 475 480agc aag gac att tca tct aag aca tgg gct gct ggt ttg aag gtt gct 1488 SerLys Asp Ile Ser Ser Lys Thr Trp Ala Ala Gly Leu Lys Val Ala 485 490 495ctt gat act ctt ttc aag tat act ccc gct cgt cct ggt gac agc act 1536 LeuAsp Thr Leu Phe Lys Tyr Thr Pro Ala Arg Pro Gly Asp Ser Thr 500 505 510atg tgt gat gct ctt gtt cca ttt gtc gaa aca ttt gtt aaa act aat 1584 MetCys Asp Ala Leu Val Pro Phe Val Glu Thr Phe Val Lys Thr Asn 515 520 525gat ctt aat gct gcc gta gag gag gct cgt aaa ggt gct gat gct act 1632 AspLeu Asn Ala Ala Val Glu Glu Ala Arg Lys Gly Ala Asp Ala Thr 530 535 540gca gat atg caa gcc aaa ctt gga cgt gct gtc tac gtt ggt gat gat 1680 AlaAsp Met Gln Ala Lys Leu Gly Arg Ala Val Tyr Val Gly Asp Asp 545 550 555560 gtt aaa gtt ccc gat gcc ggc gct ctt ggt gtc gtt gca att gtc gaa 1728Val Lys Val Pro Asp Ala Gly Ala Leu Gly Val Val Ala Ile Val Glu 565 570575 gga ttt acg aaa taa 1743 Gly Phe Thr Lys 580 2 580 PRTSchizosaccharomyces pombe 2 Met Asp Lys His Phe Ile Asn Asp Pro Glu ValLeu Val Leu Asp Gly 1 5 10 15 Leu Lys Ser Leu Ala Asp Met Asn Lys ThrLeu Thr Val His Glu Glu 20 25 30 Gly Lys Phe Ile Tyr Phe His Asp Tyr AsnLys Lys Asn Val Ser Val 35 40 45 Ile Ser Gly Gly Gly Ala Gly His Glu ProThr His Ser Ser Phe Val 50 55 60 Gly Lys Gly Met Leu Thr Ala Ala Val SerGly Ser Ile Phe Ala Ser 65 70 75 80 Pro Ser Ser Lys Gln Ile Tyr Thr GlyIle Lys Gln Val Glu Ser Glu 85 90 95 Ala Gly Thr Leu Val Ile Cys Lys AsnTyr Thr Gly Asp Ile Leu His 100 105 110 Phe Gly Met Ala Leu Glu Lys GlnArg Thr Ala Gly Lys Lys Ala Glu 115 120 125 Leu Ile Ala Val Ala Asp AspVal Ser Val Gly Arg Lys Lys Ser Gly 130 135 140 Lys Val Gly Arg Arg GlyLeu Ser Gly Thr Val Leu Val His Lys Ile 145 150 155 160 Ala Gly Ala AlaAla Ala Arg Gly Leu Pro Leu Glu Ala Val Thr Thr 165 170 175 Ile Ala LysAla Ala Ile Asp Asn Leu Val Ser Ile Gly Ala Ser Leu 180 185 190 Ala HisVal His Val Pro Gly His Glu Pro Ile Ala Lys Glu Asp Glu 195 200 205 MetLys His Asp Glu Met Glu Leu Gly Met Gly Ile His Asn Glu Pro 210 215 220Gly Cys Lys Arg Ile Ser Pro Ile Pro Ser Ile Asp Asp Leu Ile Ala 225 230235 240 Gln Met Leu Lys Gln Met Leu Asp Gln Ser Asp Lys Asp Arg Ala Tyr245 250 255 Val Lys Ile Glu Gly Asp Asp Glu Val Val Leu Leu Met Asn AsnLeu 260 265 270 Gly Gly Leu Ser Met Leu Glu Phe Ser Ala Ile Ser His LysVal Lys 275 280 285 Glu Ala Leu Ala Lys Glu Tyr Lys Ile Asn Pro Val ArgIle Phe Ala 290 295 300 Gly Pro Phe Thr Thr Ser Leu Asn Gly Leu Gly PheGly Ile Thr Leu 305 310 315 320 Leu Arg Thr Thr Asp Arg Val Lys Val GluGly Glu Glu Tyr Ser Leu 325 330 335 Val Asp Leu Ile Asp Gln Pro Val GluAla Ile Gly Trp Pro Leu Cys 340 345 350 Gln Pro Ser Asp Leu Lys Ser LysAsn Lys Ile Gly Asn Val Ser Ile 355 360 365 Glu Glu Gly Gln Lys Asp ValLys Ser Pro Val Thr Val Asp Lys Glu 370 375 380 Lys Val Arg Gln Ala IleVal Asn Ser Met Glu Asn Leu Ile Lys Ala 385 390 395 400 Glu Pro Lys IleThr Lys Phe Asp Thr Met Ala Gly Asp Gly Asp Cys 405 410 415 Gly Thr ThrLeu Lys Arg Gly Ala Glu Gly Val Leu Lys Phe Val Lys 420 425 430 Ser AspLys Phe Ser Asp Asp Pro Ile Arg Ile Val Arg Asp Ile Ala 435 440 445 AspVal Ile Glu Asp Asn Met Asp Gly Thr Ser Gly Ala Leu Tyr Ala 450 455 460Ile Phe Phe His Gly Phe Ala Lys Gly Met Lys Asp Thr Leu Glu Lys 465 470475 480 Ser Lys Asp Ile Ser Ser Lys Thr Trp Ala Ala Gly Leu Lys Val Ala485 490 495 Leu Asp Thr Leu Phe Lys Tyr Thr Pro Ala Arg Pro Gly Asp SerThr 500 505 510 Met Cys Asp Ala Leu Val Pro Phe Val Glu Thr Phe Val LysThr Asn 515 520 525 Asp Leu Asn Ala Ala Val Glu Glu Ala Arg Lys Gly AlaAsp Ala Thr 530 535 540 Ala Asp Met Gln Ala Lys Leu Gly Arg Ala Val TyrVal Gly Asp Asp 545 550 555 560 Val Lys Val Pro Asp Ala Gly Ala Leu GlyVal Val Ala Ile Val Glu 565 570 575 Gly Phe Thr Lys 580 3 33 DNAArtificial Sequence Synthetically generated primer 3 gccggtaccaggagctaaaa tatggataag cac 33 4 33 DNA Artificial Sequence Syntheticallygenerated primer 4 gccaagctta aaatttcgta tccaattatt tcg 33

What is claimed is:
 1. A method for producingdihydroxyacetone-3-phosphate, the method comprising contactingdihydroxyacetone with (a) a bacterial cell (1) transformed with anucleic acid sequence encoding a dihydroxyacetone kinase comprising theamino acid sequence set forth in SEQ ID NO:2 and (2) producing saidkinase, or (b) an extract of said bacterial cell.
 2. A method accordingto claim 1, wherein said dihydroxyacetone kinase consists of the aminoacid sequence set forth in SEQ ID NO:2.
 3. A method according to claim1, wherein said nucleic acid sequence comprises the sequence set forthin SEQ ID NO:1.
 4. A method according to claim 1, wherein said bacterialcell is an Escherichia coli cell.
 5. A method according to claim 1,wherein the contacting is carried-out in the presence of a regenerationsystem for ATP.
 6. A bacterial cell (1) transformed with a nucleic acidsequence encoding a dihydroxyacetone kinase comprising the amino acidsequence set forth in SEQ ID NO:2 and (2) producing said kinase.
 7. Thebacterial cell of claim 6, wherein said dihydroxyacetone kinase consistsof the amino acid sequence of SEQ ID NO:2.
 8. The bacterial cell ofclaim 6, wherein said nucleic acid sequence comprises the nucleotidesequence of SEQ ID NO:1.
 9. A bacterial cell lysate comprising adihydroxyacetone kinase comprising the amino acid sequence set forth inSEQ ID NO:2.
 10. The lysate of claim 9, wherein the dihydroxyacetonekinase consists of the amino acid sequence of SEQ ID NO:2.
 11. Anisolated nucleic acid comprising the sequence of SEQ ID NO: 1, or adegenerate variant of SEQ ID NO:1.
 12. A vector containing the nucleicacid of claim
 11. 13. A host cell containing the vector of claim
 12. 14.The host cell of claim 13, wherein the host cell is a bacterial cell.15. An isolated nucleic acid comprising the sequence of SEQ ID NO:1. 16.A vector containing the nucleic acid of claim
 15. 17. A host cellcontaining the vector of claim
 16. 18. The host cell of claim 17,wherein the cell is a bacterial cell.
 19. The method of claim 1, whereinthe dihydroxyacetone kinase is a Schizosaccharomyces pombedihydroxyacetone kinase.
 20. The bacterial cell of claim 6, wherein thedihydroxyacetone kinase is a Schizosaccharomyces pombe dihydroxyacetonekinase.
 21. The bacterial lysate of claim 9, wherein thedihydroxyacetone kinase is a Schizosaccharomyces pombe dihydroxyacetonekinase.